Pichia kudriavzevii and application thereof in duck tongue processing

Abstract

The invention discloses pichia kudriavzevii and application thereof in duck tongue processing, the preservation number of the pichia kudriavzevii is CGMCC No.31268, the pichia kudriavzevii is applied to duck tongue processing, and the method comprises the following steps: (1) slant culture: inoculating a slant culture medium with the pichia kudriavzevii for culture; (2) seed culture: inoculating a strain into a fresh seed culture medium for culture to obtain a seed solution; (3) preparation of fermentation liquor: taking a fermentation container, adding a saline solution, chilies and the seed solution, and carrying out fermentation culture at 15-25 DEG C for 36-72 hours; and (4) duck tongue treatment: cleaning the duck tongues, putting the cleaned duck tongues in a fermentation container, adding the pichia kudriavzevii fermentation liquor, and treating the duck tongues at 4-8 DEG C for 10-20 hours so as to complete fermentation and fishy smell removal operation. The method has the advantages that the fishy smell of the duck tongues is effectively removed, and the quality of the duck tongues is improved.

Description

Technical Field

[0001] The invention relates to the fields of microorganisms and food processing, and specifically relates to Pichia kudriavzevii yeast and application thereof in duck tongue processing. Background Art

[0002] Duck tongue is a by-product of duck processing. It is rich in cartilage tissue, the tip of the tongue is crisp, soft and smooth, the protein content is high, and it is rich in trace elements such as phosphorus, calcium, iron, zinc, and selenium. It is not only fresh and mellow, but also has the effects of nourishing the stomach, nourishing yin, replenishing blood, and promoting salivation. It is a nutritious and convenient new type of cold dish and snack food. The consumption is increasing day by day, and the market prospects are broad.

[0003] Duck tongue is rich in nutrition and delicious, but it has a strong duck smell. Improper handling will affect consumer acceptance and the development of the industry. At present, people have higher and higher requirements for food flavor. How to control the formation of fishy substances and remove fishy substances has attracted more and more attention from researchers. At present, the main fishy removal technologies for livestock and poultry by-products include masking method, adsorption method, embedding / microencapsulation method, acid-base salt treatment method, natural antioxidant method, oxidant method, Maillard reaction method, microbial fermentation method, etc. (Liu Zeqi, Li Mingxia, Yang Lin, Zheng Hua, Lin Jie, Wu Shaozong, Guo Zonglin, Lei Hongtao. Research progress on the formation mechanism and removal methods of fishy smell of livestock and poultry by-products [J]. Journal of Food Safety and Quality, 2023, 14(16): 86-93). For duck tongue, spices, soy sauce, liquor and other substances are mainly used to cover and remove the fishy smell. CN105876455A discloses a method for processing duck tongue with sauce flavor, which uses a liquid prepared by brewing soy sauce, liquor, star anise, cinnamon, grass fruit, five-spice powder, thirteen-spice powder, ginger, green onion, etc. to soak and improve the flavor of duck tongue; CN103181571A discloses a method for processing duck tongue with seasonings such as soy sauce, bean paste, balsamic vinegar, star anise, cinnamon, bay leaf, ginger, cumin, thyme, angelica, etc.; CN102342530A discloses a method for processing duck tongue with spices such as star anise, cinnamon, prickly ash, white pepper, ginger, cardamom, cloves, etc. The masking method can remove the fishy smell of duck tongue to a certain extent, but it requires many types of seasonings / spices and cannot fundamentally change the presence of fishy substances.

[0004] Microbial deodorization is a green and environmentally friendly deodorization method, which mainly relies on microbial metabolism or microbial enzymes to convert fishy substances into odorless substances, and can fundamentally remove or change fishy substances with good results. However, there are currently no microorganisms that can be well applied to duck tongue processing. Summary of the invention

[0005] The invention aims to provide a Pichia kudriavzevii yeast and application thereof in duck tongue processing.

[0006] To achieve the above object, the present invention provides a Pichia kudriavzevii, whose preservation number is: CGMCC No.31268.

[0007] The Pichia kudriavzevii of the present invention has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on December 4, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The strain name of the strain is: WZ-2024-12; the classification name is Pichia kudriavzevii; the deposit number is: CGMCC No. 31268. The nucleotide sequence of the Pichia kudriavzevii is shown in SEQ ID NO: 1.

[0008] The invention also provides an application of the Pichia kudriavzevii in duck tongue processing.

[0009] Further setup is to include the following steps:

[0010] (1) Slant culture: inoculating the Pichia kudriavzevii onto a slant culture medium;

[0011] (2) Seed culture: The bacteria grown on the slant medium are inoculated into fresh seed culture medium to obtain a Pichia kudriavzevii seed solution;

[0012] (3) Preparation of fermentation liquid: taking a fermentation container, adding 5-10wt% of salt water, 10-20wt% of pepper, and 1-5wt% of the Pichia kudriavzevii seed liquid, and fermenting and culturing at 15-25°C for 36-72 hours to obtain Pichia kudriavzevii fermentation liquid;

[0013] (4) Duck tongue processing: The duck tongue is cleaned and placed in a fermentation container. Pichia kudriavzevii fermentation liquid is added and processed at 4-8°C for 10-20 hours to complete the fermentation and deodorization operation.

[0014] The further configuration is that the slant culture medium and seed culture medium are MRS culture medium.

[0015] The positive and progressive effects of the present invention are:

[0016] After the Pichia kudriavzevii provided by the present invention is fermented with duck tongue, the fishy smell of duck tongue can be removed effectively. The method of the present invention has the advantages of simple process, low cost, good fishy smell removal effect on duck tongue, and after being treated by the method of the present invention, the meat texture of the product can be improved, the chewiness is good, the flavor is fragrant, and it is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0018] Figure 1 Colony image of Pichia kudriavzevii CGMCC No.31268;

[0019] Figure 2 GC-MS total ion chromatogram of volatile components of duck tongue;

[0020] Figure 3 GC-MS total ion chromatogram of volatile components of duck tongue fermented with Pichia kudriavzevii CGMCC No.31268. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0023] The bacterial culture medium used in the following examples is as follows:

[0024] Strain screening medium (g / L): peptone 10, beef extract 5, yeast extract 5, glucose 2, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05,

[0025] Agar 20, pH 6.8, streptomycin 500U;

[0026] Slant medium (g / L): peptone 10, beef extract 5, yeast extract 5, glucose 2, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, magnesium sulfate 0.2, manganese sulfate 0.05, agar 20, pH 6.8;

[0027] Seed culture medium (g / L): peptone 10, beef extract 5, yeast extract 5, glucose 2, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, magnesium sulfate 0.2, manganese sulfate 0.05, pH 6.8.

[0028] Example 1 Screening, Isolation and Identification of Pichia kudriavzevii WZ-2024-12

[0029] 1. Pichia kudriavzevii WZ-2024-12 is isolated from fermented vegetables. Take the fermented vegetable liquid and dilute it by 10 -5 , 10 -6 , 10 -7 Times, take 100 μl and spread it on the strain screening medium plate, repeat three times for each concentration, culture at 32℃ for 48 hours, select the round and slightly flat white colonies, and purify them several times to obtain 16 strains, inoculate the purified strains on the slant medium, culture at 32℃ for 24 hours, and then store them in a 4℃ refrigerator for use.

[0030] The strain stored in a refrigerator at 4°C was inoculated into the seed culture medium. After culturing at 32°C for 24 hours, 10 ml of seed liquid was inoculated into 500 g of brine (10% salt content) containing 100 g of pepper. The mixture was fermented at 25°C for 72 hours. Duck tongue was then added in a 1:1 ratio for 12 hours. The results of sensory evaluation are shown in Table 1. It can be seen from Table 1 that strain No. 12 has the strongest deodorizing effect and is named WZ-2024-12.

[0031] Table 1 Effect of strains screened on deodorizing duck tongue

[0032]

[0033]

[0034] 2. Molecular biological identification of Pichia kudriavzevii WZ-2024-12

[0035] The target strain was cultured on a slant medium for 24 hours, genomic DNA was extracted, and 18S rDNA-ITS of the strain was amplified by PCR. The primers used were:

[0036] NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3', as shown in SEQ ID NO: 2)

[0037] NL4 (5'-GGTCCGTGTTTCAAGACGG-3', as shown in SEQ ID NO: 3);

[0038] The composition of the PCR reaction system is as follows:

[0039]

[0040]

[0041] The PCR conditions are shown in the following table:

[0042]

[0043] The PCR product was sequenced and compared with the 18S rDNA-ITS in the GeneBank database. The results are shown in Table 2. It was found that the similarity with strains such as Pichia kudriavzevii solate 214 (GenBank accession No. PP059503.1) was as high as 100%, and the strain was determined to be Pichia kudriavzevii.

[0044] Table 2 Sequence of strain WZ-2024-12 and the 10 most similar strains in GenBank

[0045]

[0046]

[0047] Example 2 GC-MS analysis of volatile components of duck tongue

[0048] (1) Solid Phase Microextraction

[0049] Duck tongue was homogenized with a crusher; 5 g of sample and 5 μL of internal standard toluene-D8 were placed in a 20 mL headspace vial. The vial was incubated in a 60°C water bath for 20 minutes, and then an extraction needle (Supelco, DVB / CAR / PDMS, 50 / 30 μm) was inserted into the headspace vial for flavor enrichment, and the enrichment time was 60 minutes.

[0050] (2) Instruments and measurement conditions

[0051] Agilent gas chromatograph-mass spectrometer GC 6890-MS5975.

[0052] Chromatographic conditions: Agilent 6890N gas chromatograph, DB-5 capillary column (60m×250μm, 0.25μm). The column temperature was programmed to rise, initially at 35°C for 6 min, then raised to 80°C at 5°C / min, held for 2 min, and then raised to 230°C at 6°C / min. Splitless injection was used, with an injection port temperature of 270°C; the carrier gas was He, with a flow rate of 1.0 mL / min.

[0053] Mass spectrometry conditions: Agilent 5975MSD mass spectrometer, electron impact ion source (EI), ion source temperature 230°C, interface temperature 280°C, scanning mass range 10amu-500amu. Environmental conditions: room temperature 25°C.

[0054] (3) Data processing

[0055] The Nist standard spectral library of the Agilent G1701 MSD Productivity Chem Station enhanced data analysis workstation was used to automatically retrieve the mass spectrometry data of each component, and the machine inspection results were checked and confirmed according to the standard spectrum and retention index. The content of each component was semi-quantitatively determined based on the ratio of the peak area to the peak area of ​​the internal standard (toluene).

[0056] Example 3

[0057] Inoculate Pichia kudriavzewei WZ-2024-12 onto a slant culture medium and culture at 32°C for 24 hours; then inoculate the strain grown on the slant culture medium into a fresh seed culture medium, culture at 32°C for 24 hours to obtain Pichia kudriavzewei seed solution; take a fermentation container, add 10wt% of salt water, 10wt% of chili pepper, and 2wt% of the Pichia kudriavzewei seed solution, ferment and culture at 20°C for 72 hours to obtain Pichia kudriavzewei fermentation liquid; clean the duck tongue, place it in a fermentation container, add Pichia kudriavzewei fermentation liquid at a ratio of 1:1, and treat at 5°C for 15 hours. Take the duck tongue before and after fermentation and perform volatile component detection according to the method of Example 2. The total ion flow diagrams of the volatile components of the duck tongue raw material and the duck tongue fermented with Pichia kudriavzewei are shown as follows: Figure 2 , Figure 3 As shown in Table 3 and Table 4, the volatile components and their contents are respectively detected in Table 3 and Table 4. It can be seen from the results that 19 volatile components were detected in duck tongue raw materials, mainly aldehydes and enals; after fermentation with Pichia kudriavzevii, the types of volatile components increased significantly to 47, and the aldehydes and enals were oxidized and reduced to other components during the fermentation process, and the types and contents were significantly reduced. Some alcohols, esters and acids were produced by metabolism, which changed the composition of volatile components and eliminated the fishy smell.

[0058] Table 3 Main volatile components of duck tongue raw materials

[0059]

[0060] Table 4 Volatile components of duck tongue fermented by Pichia kudriavzevii WZ-2024-12

[0061]

[0062]

[0063] Example 4

[0064] The activation and seed liquid preparation of Pichia kudriazevich WZ-2024-12 are the same as those in Example 3. Take a fermentation container, add 7wt% of saline, 15wt% of pepper, and 2wt% of the Pichia kudriazevich seed liquid, and ferment and culture at 25°C for 30 hours to obtain Pichia kudriazevich fermentation liquid; wash the duck tongue, place it in a fermentation container, add Pichia kudriazevich fermentation liquid at a ratio of 1:1, and treat it at 4°C for 20 hours. After the fermentation, the volatile components of the duck tongue were analyzed by GC-MS, and the results similar to those in Example 3 were achieved. The duck tongue fermented with Pichia kudriazevich had no fishy smell.

[0065] Example 5

[0066] The activation and seed liquid preparation of Pichia kudriazevich WZ-2024-12 are the same as those in Example 3. Take a fermentation container, add 10wt% of salt water, 20wt% of pepper, and 3wt% of the Pichia kudriazevich seed liquid, and ferment and culture at 15°C for 72 hours to obtain Pichia kudriazevich fermentation liquid; wash the duck tongue, place it in a fermentation container, add Pichia kudriazevich fermentation liquid at a ratio of 1:1, and treat it at 4°C for 15 hours. After the fermentation, the volatile components of the duck tongue were analyzed by GC-MS, and the results similar to those in Example 3 were achieved. The duck tongue fermented with Pichia kudriazevich had no fishy smell.

[0067] Example 6

[0068] The activation and seed liquid preparation of Pichia kudriazevich WZ-2024-12 are the same as those in Example 3. Take a fermentation container, add 7wt% of saline, 20wt% of pepper, and 5wt% of the Pichia kudriazevich seed liquid, and ferment and culture at 20°C for 48 hours to obtain Pichia kudriazevich fermentation liquid; wash the duck tongue, place it in a fermentation container, add Pichia kudriazevich fermentation liquid at a ratio of 1:1, and treat it at 8°C for 10 hours. After the fermentation, the volatile components of the duck tongue were analyzed by GC-MS, and the results similar to those in Example 3 were achieved. The duck tongue fermented with Pichia kudriazevich had no fishy smell.

[0069] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A Pichia kudriavzevii yeast having a function of removing the fishy smell of duck tongue, and its deposit number is: CGMCC No.31268.

2. Use of the Pichia kudriavzevii according to claim 1 in duck tongue processing.

3. The use according to claim 2, characterized in that The following steps are involved: (1) Slant culture: inoculating the Pichia kudriavzevii onto a slant medium for culture; (2) Seed culture: The bacteria grown on the slant medium are inoculated into fresh seed culture medium to obtain a Pichia kudriavzevii seed solution; (3) Preparation of fermentation liquid: taking a fermentation container, adding 5-10wt% of salt water, 10-20wt% of pepper, and 1-5wt% of the Pichia kudriavzevii seed liquid, and fermenting and culturing at 15-25°C for 36-72 hours to obtain Pichia kudriavzevii fermentation liquid; (4) Duck tongue processing: The duck tongue is cleaned and placed in a fermentation container. Pichia kudriavzevii fermentation liquid is added and processed at 4-8°C for 10-20 hours to complete the fermentation and deodorization operation.

4. The use according to claim 3, characterized in that: The slant culture medium and seed culture medium are MRS culture medium.

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